Michael Bárány

Proton magnetic resonance spectroscopy of the brain in schizophrenic and affective patients

Water-suppressed 1H magnetic resonance spectra were recorded from two brain regions of psychiatric patients and normal volunteers. The two regions studied were (a) the basal ganglia structures surrounding the anterior horn of the lateral ventricle and (b) the occipital cortex. N-Acetylaspartate (NAA), phosphocreatine-creatine (PCr-Cr), choline and inositol resonances were seen in both regions. Ratios of metabolite peak integrals to PCr-Cr peak integral were calculated for each spectrum. To control for partial volume effects, comparisons between patients and controls were made only from identical regions i.e. basal ganglia vs basal ganglia, and likewise for occipital cortex. Metabolite ratios from the occipital region of patients were similar to those from the occipital region of normal subjects. Bipolar patients being treated with lithium had elevated NAA/PCr-Cr in the basal ganglia region when compared to normals. These patients also demonstrated elevated choline/PCr-Cr and inositol/PCr-Cr ratios in the basal ganglia region.

[53] Phosphorus-31 nuclear magnetic resonance of contractile systems

Publisher Summary This chapter focuses on techniques and procedures for recording and evaluating the 31 P nuclear magnetic resonance (NMR) spectra of intact muscles and of perchloric acid extracts of muscle. At the present time, high-resolution NMR is probably the best general-purpose analytical tool available for studying the chemistry of phosphorus and its compounds. With a 31 P NMR probe in a highresolution NMR spectrometer, the analyst can determine easily, and usually within a few minutes, whether phosphorus is present at concentrations greater than 100 μM in fluid samples, characterize each type of phosphorus-containing group present, and obtain a quantitative analysis of the various species present. For a spectrometer in which a magnetic field of 23 kiloGauss is employed so that the resonance position of protons is 100 MHz, the 31 P resonance are found at 40.5 MHz; in a magnetic field 3.6 times this, the resonance will be found at 145.8 MHz.

Chicken gizzard myosin.

Abstract The Ca++-ATPase activity of chicken gizzard myosin is increased 2- to 3-fold by tryptic treatment and by various substances, urea, guanidine-HCl, sodium dodecylsulfate, KCNS, LiBr, ethanol, or ethylene glycol. These substances also activate the Ca++-ATPase of rabbit uterus myosin but have no effect on the Ca++-ATPase of chicken breast myosin. Maximal Ca++-ATPase activity of gizzard myosin is observed when 38 free NH2-terminal groups in 600,000 gm of this myosin are liberated by trypsin. At this stage most of the myosin remains intact in the analytical ultracentrifuge. The addition of 1.0–2.0 M urea to gizzard myosin, pH 7.0, results in a 3-fold decrease in the Michaelis constant. The effect of urea on the activation of Ca++-ATPase of gizzard myosin is reversible. Twelve cysteine residues per 600,000 gm of gizzard myosin are reacting with iodoacetate in the presence of 1.5 M urea in contrast to the nonreactivity of the SH groups without urea. Gizzard myosin retains all of its Ca++-ATPase activity after 12 of its SH groups have been alkylated. From comparative experiments with iodoacetate and iodoacetamide it is concluded that the ATPase site of gizzard myosin undergoes an alteration in the presence of urea as a result of conformational changes in regions of the myosin molecule removed from the ATPase site. The Ca++-ATPase and NTPase activities of gizzard myosin are much lower than those of breast myosin even in the presence of modifiers. However, the ability of the two different types of myosin to combine with rabbit skeletal F-actin is the same. Amino acid analyses show differences in the composition of gizzard and breast myosin. Gizzard myosin contains cystine and has a slightly lower cysteine content than does breast myosin.

PHOSPHODIESTERS IN MUSCULAR DYSTROPHIES

Phosphorus nuclear magnetic resonance studies of various normal and dystrophic muscles have demonstrated differences in phosphodiester contents. Dystrophic chicken pectoralis muscle contains elevated levels of the diester SEP, and Duchenne dystrophic human leg muscle lacks the diester GPC. It is shown that SEP may be characteristic of slow fiber types, indicating imcomplete maturation of the dystrophic pectoralis muscle. The activities of SEP-metabolizing enzymes have been determined in microsomes of pectoralis muscle, kidney, and intestinal mucosa of normal and dystrophic chickens. There was little difference between normal and dystrophic values of SEP phosphodiesterase and SEP synthase in kidney and mucosa. Dystrophic pectoralis muscle microsomes possessed significantly elevated levels of both enzymes. The increase in SEP synthase activity of dystrophic muscle may explain the increase in the SEP level if the phosphodiesterase is regulated in vivo. It is shown that human muscle disease processes may raise, leave unchanged, or lower GPC levels. In certain diseases when GPC is markedly elevated, such as Werdnig-Hoffmann, or depressed, such as Duchenne muscular dystrophy, it may serve as a marker.

Studies on the low molecular weight protein components in rabbit skeletal myosin

Abstract Myosin and heavy meromyosin, twice-chromatographed and free from 5′-AMP deaminase, adenylate kinase, and nucleic acids (procedure of Harris and Suelter, Ref. 20), have been found to contain low molecular weight protein components. The content of these components in twice-chromatographed myosin was found to be 4.8% of the myosin by weight. Chromatography on DEAE-Sephadex A-50 or electrophoresis on polyacrylamide gel separates these small proteins into three main components. Separation of the low molecular weight proteins from myosin, under various conditions, resulted in a concomitant loss of its ATPase activity and actin-binding ability. Addition of the isolated low molecular weight proteins to myosin, free of these proteins, did not restore its ATPase activity. The data indicate that the low molecular weight proteins are tightly bound to myosin, but do not reveal their role in the myosin molecule.

Identification of an unknown 31P nuclear magnetic resonance from dystrophic chicken as l-serine ethanolamine phosphodiester

Abstract A phosphodiester, which comes into resonance at 0.4 ppm in the 31 P nuclear magnetic resonance spectrum of intact muscles, has been isolated from the pectoralis muscle of chickens with hereditary muscular dystrophy by perchloric acid extraction, barium and alcoholic fractionation, and chromatographic isolation procedures. The compound, l -serine ethanolamine phosphodiester, whose presence is a characteristic of the diseased chicken muscle, has been characterized by 31 P, 13 C, and 1 H nuclear magnetic resonance as well as by chemical and chromatographic procedures.

Exchange of the actin-bound nucleotide in intact arterial smooth muscle.

The actin-bound ADP was separated from cytoplasmic nucleotides by treatment of intact arterial smooth muscle with 50% ethanol. In 32P-labeled smooth muscle the actin-bound ADP and phosphate readily exchanged with the cytoplasmic [γ,β-32P]ATP; the specific radioactivity of actin-bound ADP was equal to that of the β-phosphate of cytoplasmic ATP and the specific radioactivity of actin-bound phosphate was equal to that of the γ-phosphate of cytoplasmic ATP. In contrast, the exchange of the actin-bound ADP in skeletal muscle was very slow. The presence of cytoplasmic ATP was required for the exchange of the actin-bound ADP and phosphate; if ATP synthesis was inhibited the exchange was also inhibited. The extent of exchange was reduced in muscles contracted by histamine or K+, as compared with resting muscles. The exchange was also shown in other mammalian smooth muscles, uterus, urinary bladder, and stomach. The data indicate a dynamic state of actin in smooth muscle. The data also suggest that polymerization-depolymerization of actin is part of the contraction-relaxation cycle of smooth muscle.

1H NMR of intact muscle at 11 T

1H NMR spectra of intact frog, and chicken skeletal muscles, were recorded at 470 MHz with the Plateau and Guéron pulse sequence for the suppression of water [(1982) J. Am. Chem. Soc. 104, 7310]. Only a few transients were required to resolve the resonances from the protons of muscle metabolites. The previously unobserved exchangeable protons of muscles were also recorded and thereby phosphocreatine and creatine could be measured simultaneously. During aging of dissected frog muscle, changes in levels of phosphocreatine, creatine and lactic acid, and the decrease in the intracellular pH were followed by 1H NMR.

Absence of calponin phosphorylation in contracting or resting arterial smooth muscle.

We have tested the hypothesis of Winder and Walsh [(1990) J. Biol. Chem. 256, 10148] that the contractile state of smooth muscle is regulated by calponin phosphorylation. Porcine carotid arterial muscles were highly labeled with 33P, then contracted with four different agents for various times. No radioactivity was detected in calponin isolated by 2D or 1D gel electrophoresis from the muscles. Similarly, resting muscles showed no [33P]phosphate in calponin. Apparently the sites of calponin available for phosphorylation in vitro are rendered unavailable in the intact muscle.

Myosin light chain isoforms and their phosphorylation in arterial smooth muscle.

Arterial smooth muscle myosin contains nonphosphorylated and phosphorylated light chains that appear as 4 spots on two-dimensional, Coomassie blue-stained gel electrophoretograms at the 20,000-molecular weight level (referred to as spots 4 through 1 in order of decreasing isoelectric points). Anti-light chain recognizes the proteins in all 4 light chain spots. Complete dephosphorylation of light chain in muscle homogenate, by inhibiting myosin light chain kinase and by adding phosphatase, leads to 2 spots on two-dimensional gel electrophoretograms; both spots are visible on immunoblots. Stimulation (K+ or stretch) of smooth muscle results in increased light chain phosphorylation. Autoradiography of the gel electrophoretograms reveals that radioactive components are contained in spots 3, 2, 1, and in an additional spot with lower isoelectric point, referred to as spot 0. Phosphoamino acid analysis shows that spots 3 and 1 contain phosphoserine, whereas spots 2 and 0 contain phosphoserine and phosphothreonine. Two-dimensional phosphopeptide mapping of the trypsin-digested proteins from spots 3 and 1 shows predominantly 2 peptides; whereas from spots 2 and 0, it shows 5 peptides. Sodium dodecyl sulfate gel electrophoresis of the phosphopeptides obtained with Staphylococcus aureus V8 digestion gives identical maps for spots 3 and 2, which are different from the identical maps of spots 1 and 0. The results suggest that arterial smooth muscle myosin contains 2 nonphosphorylated 20,000-dalton light chain isoforms with different amino acid sequences and that each isoform can be mono- and diphosphorylated.